Integrins are transmembrane proteins that mediate interactions between adhesion molecules on adjacent cells and/or the extracellular matrix (ECM). Integrins play diverse roles in several biological processes including cell migration during development and wound healing, cell differentiation and apoptosis. Their activities can also regulate the metastatic and invasive potential of tumor cells. They exist as heterodimers consisting of α and β subunits. Some α and β subunits exhibit specificity for one another and may be designated as a VLA (very late antigen) member. Heterodimers often preferentially bind certain cell adhesion molecules, or constituents of the ECM. Although they have no catalytic activity, integrins can be part of multimolecular signaling complexes known as focal adhesions.
Upon binding to ligands, integrins transduce intracellular signals to the cytoskeleton that modify cellular activity in response to these cellular adhesion events, referred to as outside-in signaling. Such signaling can also activate other integrin subtypes expressed on the same cell, referred to as inside-out signaling. Inside-out signaling further occurs via regulatory signals that originate within cell cytoplasm such as a disruption of the clasp between an α and β subunit, which are then transmitted to the external ligand-binding domain of the receptor. Integrins can play important roles in the cell adhesion events that control development, organ morphogenesis, physiology and pathology as well as normal tissue homeostasis, and immune and thrombotic responses, and in addition, they serve as environmental sensors for the cell.
One of the integrin heterodimers is α2β1 integrin. The α2β1 integrin is expressed on several different cell types, including endothelial and epithelial cells, fibroblasts, lymphocytes, and platelets. The ligand specificity of α2β1 varies with cell type. While it serves as a collagen receptor on platelets and fibroblasts, it can serve as both a collagen and as a laminin receptor on endothelial and epithelial cells.
α2β1 integrin is a molecule composed of an α2 integrin subunit of the family of a integrins, and a β1 integrin subunit from the family of β integrins. The sequences of α2 and β1 integrin are known in the art and are published, e.g. in Takada and Hemler J. Cell Biol. 109(1):397-407, 1989 and Argraves, W. S, J. Cell. Biol. September 105(3): 1183-90 (1987). Example sequences are denoted in FIG. 9 and further sequences can be retrieved from the National Centre for Biotechnology Information (NCBI) data base, e.g. under NCBI accession Numbers NP—002194 NM—002203, NM—002211, NP—002202 (β1 integrin isoform 1A) for homo sapiens α2 and β1 integrin, see also below.
Alternative splice variants, isoforms are known in the art, as well as sequences of non-human origin (such as rodent—mouse, rat, etc—simian or other) and represent possible alternative embodiments as long as they exhibit at least one of the known functions of α2 or β1 integrin.
The α2 subunit is a member of a subset of integrin α subunits that contain an approximately 200 amino acid domain located near the amino terminus often referred to as the I (or inserted) domain. Many I domains, including the α2 and integrin subunit I domain, contain an additional cation binding site, the metal ion-dependent adhesion site (MIDAS) motif. The structural characterisation of the α2 integrin I domain is published, e.g. in Dickeson et. al., J. Biol. Chemistry, 272, 7661-7668 (1997). I domains are important determinants in ligand binding. The amino acid sequence of a human α2 integrin I domain can be gained from FIG. 9, as marked in the α2 integrin sequence (SEQ ID 20).
The α2β1 integrin (very late antigen 2; VLA-2) is expressed on a variety of cell types including platelets, vascular endothelial cells, epithelial cells, activated monocytes/macrophages, fibroblasts, leukocytes, lymphocytes, activated neutrophils and mast cells. The natural ligands for α2β1 include collagen and laminin, both of which are found in extracellular matrix. The α2β1 integrin has been implicated in several biological and pathological processes including collagen-induced platelet aggregation, cell migration on collagen, cell-dependent reorganization of collagen fibers as well as collagen-dependent cellular responses that result in increases in cytokine expression and proliferation, aspects of T-cell, mast cell, and neutrophil function, aspects of delayed type hypersensitivity contact hypersensitivity and collagen-induced arthritis, mammary gland ductal morphogenesis, epidermal wound healing, and processes associated with VEGF-induced angiogenesis.
Platelets normally circulate in the blood in an inactive resting state, however, they are primed to respond rapidly at sites of injury to a wide variety of agonists. Upon stimulation, they undergo shape changes and become highly reactive with plasma proteins, such as fibrinogen and von Willebrand factor (vWf), other platelets, and the endothelial lining of the vessel wall. These interactions all cooperate to facilitate the rapid formation of a hemostatic fibrin platelet plug (Cramer, 2002 in Hemostasis and Thrombosis, 4th edition). Upon binding ligand, platelet receptors transduce outside-in signal pathways which in turn, trigger inside-out signaling that results in activation of secondary receptors such as the platelet fibrinogen receptor, αIIbβ3 integrin, leading to platelet aggregation. Even minor activation of platelets can result in platelet thrombotic responses, thrombocytopenia and bleeding complications.
α2 integrin is the only collagen-binding integrin expressed on platelets and has been implicated to play some role in platelet adhesion to collagen and hemostasis (Santoro et al., Thromb. Haemost. 74:813-821 (1995); Vanhoorelbeke et al., Curr Drug Targets Cardiovasc. Haematol. Disord. 3(2): 125-40 (2003); Sarratt et al., Blood 106(4): 1268-1277 (2005)). Therefore, the inactivation of alpha 2 integrin function would be desirable in order to negatively interfere with platelet aggregation. One such kind of inhibition would e.g. be an allosteric inhibition that locks the integrin in the inactive state.
Integrin/ligand interactions can facilitate leukocyte extravasations into inflamed tissues (Jackson et al., J. Med. Chem. 40:3359-3368 (1997); Gadek et al., Science 295(5557):1086-9 (2002), Sircar et al., Bioorg. Med. Chem. 10:2051-2066 (2002)), and play a role in downstream events following the initial extravasations of leukocytes from the circulation into tissues in response to inflammatory stimuli, including migration, recruitment and activation of pro-inflammatory cells at the site of inflammation (Eble J. A., Curr Pharm Des. 11(7):867-880 (2005)).
Blocking of α2 integrin has been reported to show impact on delayed hypersensitivity responses and efficacy in a murine model of rheumatoid arthritis and a model of inflammatory bowel disease (Kriegelstein et al., J. Clin. Invest. 110(12):1773-82 (2002); de Fougerolles et al., J. Clin. Invest. 105:721-720 (2000) and attenuate endothelial cell proliferation and migration in vitro (Senger et al., Am. J. Pathol. 160(1):195-204 (2002), suggesting that the blocking of α2 integrin might prevent/inhibit abnormal or higher than normal angiogenesis, as observed in various cancers. Furthermore, in a rat colorectal cancer surgery model α2-integrin inhibition was shown to be an effective anti-metastatic (van der Bji et al, Hepatology 47(2): 532-543 (2008)). Lineage commitment of colorectal cancer cells could also be shifted away from malignant phenotype (Kirkland et al J Biol Chem 283(41): 27612-27619 (2008)). As a 2 integrin was shown to mediate the malignant phenotype in pancreatic cancer (Grzesiak and Bouvet, Br J Cancer 94: 1311-1319 (2006) validating this target for a therapeutic approach in this type of aggressive cancer. Moreover, α201 integrin is interacting with glycosphingolipids in the progression of prostate cancer suggesting that blockade of this interaction will be of therapeutic use for this type of cancer (van Slambrouck et al., Int J Onco 35: 693-699 (2009). In experimental autoimmune encephalitis (EAE), a murine model of multiple sclerosis (MS), α2 integrin seems to play an important role as treatment with an anti-α2 antibody, given immediately after the onset of the disease, suppressed clinical signs and inflammation of the CNS (Tsunoda et al Brain Pathol 17:45-55 (2007). The mechanism of this therapeutically beneficial action of the anti-α2 antibody is most likely due to the inhibition of the interaction of α2β1 integrin with C1q complement protein. This interaction is a first step in mast-cell-degranulation and mast-cell activation, which is involved in autoimmune and inflammatory diseases, like MS, systemic lupus erythematosus, glomerolonephritis (McCall-Culbreath et al Blood 111(3562-3570) 2008).
Thus, α2 integrin is an interesting medical target. As integrins are difficult targets for the development of specific inhibitors, and in view of the many different possible therapeutic indications, there is a need for alternative inhibitors binding to α2 integrin, especially inhibitors of alpha 2 integrin exhibiting somewhat different properties when compared with existing α2 integrin inhibitors, which can be used in the treatment of α2 integrin-associated disorders.